Tissue engineering involves the delivery of scaffold materials and/or living cells to repair and/or replace damaged, degenerative or dysfunctional tissues. Tissue engineered implants may be used for the clinical repair or replacement of damaged or degenerative tissues of a variety of tissues/organs.
Typically, tissue engineered implants comprise a scaffold material (either synthetic or biological in origin) which can be delivered as an acellular matrix that promotes or guides tissue regeneration or, as a ‘living’ composite, which has been seeded in vitro with viable cells prior to implantation. Ideally, the cells used in these ‘living’ implants have been isolated from the graft recipient's own tissue (autogeneic), however ‘banked’ cells from different donors (allogeneic) may also be used.
One approach to tissue engineering has been to construct tissue implants in vitro, starting from their component matrix and cellular elements, with or without the inclusion of a synthetic polymer scaffold, which may be required in order to provide mechanical strength during the initial period of matrix synthesis, reorganisation and maturation. The pre-formed architecture of natural tissue matrices offers a number of advantages over the use of synthetic materials and laboratory generated biological matrices. Primarily, implants engineered using natural matrices can more closely reproduce the biomechanical and functional properties of the replaced tissue without the need for mechanical ‘pre-conditioning’. Natural matrices may be derived from either human (allogeneic) or non-human species (xenogeneic). Regardless of their source, they are frequently decellularised in order to reduce their immunogenicity. It is known in the prior art, as described in EP 0773033, to use an ultrasonic wash/bath at a low energy intensity to clean away/remove cellular layers from connective tissue membranes. Additionally, decellularisation can be achieved by biochemical modification using detergents, or enzymes. Additionally, cross-linking the component polymers is used to enhance their stability, and hence their longevity, in vivo. A problem associated with biochemically modified matrices is their resistance to rapid recellularisation. Biochemical modification such as cross-linking can render the tissue environment more hostile to infiltrating cells and hence delay recellularisation, a process that is considered essential for the restoration of biological function.
In an attempt to improve recellularisation, it is known from the prior art to coat the surface of the matrix with beneficial molecules, such as growth factors or adhesion molecules that promote cell attachment. However, even after coating the matrix surface there is a problem with encouraging the colonising cells to penetrate into the depths of the tissue so that they begin to remodel the existing structure and synthesise new extracellular matrix. Previous approaches to this problem (which affects cellular infiltration both in vitro and in vivo) have included mechanical or laser ‘punching’ of holes in the structure in a fine array. However, neither of these techniques are able to produce a continuous effect and they only allow cellular access to the region surrounding the holes and not the whole of the matrix. It is also known from the prior art to use jet injection technology to propel cells at a three dimensional matrix such that the cells become embedded within the interior of the matrix. This method is described in WO96/40889. However, the problem with this method is that the cells can become mechanically damaged due to the jet propulsion and eventually necrose.
A further attempt to improve recellularisation lies in the use of ice crystals which are formed during repeated freeze thawing or freeze-drying, the ice crystals may be used to disrupt the collagenous network. A disadvantage of this method is that the resultant physical changes are not only difficult to control and but can produce significant deterioration in the mechanical properties of treated tissues.
The present invention provides a novel technique which mitigates some of the prior art problems.